The over-all objective of this project is identifying and developing selective nontoxic inhibitors of the hepatitis C virus NS5B polymerase and eventually testing them as therapeutics for chronic hepatitis C. At least four million people in the United States have chronic hepatitis C and the recent NIH Consensus Conference suggested that this number is actually two-three fold greater. The high rate of progression to chronic infection (70-80%), liver disease (>50%), and the worldwide distribution of chronic hepatitis C, makes it a major cause of morbidity and mortality. The production of HCV vaccines is a difficult challenge and an intensive development of molecular based HCV therapeutics should be pursued. A major problem in HCV research has been the development of model systems to study HCV replication and identifying effective molecular based therapeutics. We have developed methods for expressing in E. coli and purifying mg quantities of purified enzymatically active NS5B polymerase protein. In addition, crystal structures of NS5B polymerase have been solved and robust cell based replicon systems can be used to test for inhibition of HCV RNA replication. These developments permit virtual screening of small molecules as candidate NS5B polymerase inhibitors. The specific objectives of this project are: 1) to express and purify sufficient quantities of recombinant HCV NS5B polymerase for in vitro assays and crystal growth studies; 2) To test additional compounds, already identified by structure-based virtual screening, for their ability to directly inhibit the HCV NS5B polymerase using an in vitro assay. These results and current NS5B structural information will be used to further design, synthesize and test modifications of lead compounds; 3) To conduct co-crystal growth studies of NS5B, a template, and an inhibitor with the goal of using this ternary complex structure to design and optimize NS5B polymerase inhibitors.